Modulation of Wnt5a to Treat Glaucoma

ABSTRACT

Glaucoma or pathogenic intraocular pressure is treated by locally administering to an eye in need thereof formulations of a Wnt5a inhibitor.

This invention was made with government support under Grant NumbersEY017392 and EY028995 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

INTRODUCTION

Glaucoma is a major health problem which affects over 3 millionAmericans and 60 million people worldwide. It is estimated that 111.8million people will be affected by this disease worldwide in 2040. Amajor risk factor for this disease is increased intraocular pressure(IOP), which can damage the optic nerve and cause permanent blindnesswithout treatment. Currently, there is no cure for glaucoma. Existingeye drops or oral medications are of limited efficacy with many sideeffects, and surgeries often fail with scar formation and fibrosis.

Aqueous humor is the clear colorless liquid that fills the anterior andposterior chambers of the eye. It is produced by the ciliary body at theposterior chamber and exits the anterior chamber angle through theconventional pathway via trabecular meshwork and Schlemm's canal, andthe nonconventional pathway via uveoscleral outflow. In normal eyes, adynamic balance exists between the production and drainage of aqueoushumor, maintaining IOP in the normal range.

Schlemm's canal (SC) is a circumferential channel located at theiridocorneal angle in the ocular anterior chamber. It is part of theconventional aqueous humor outflow system, which accounts for 70-90% ofthe total aqueous humor that drains out of the eye in human. Theendothelial cell lining of Schlemm's canal is one of the primary sitesof resistance to aqueous humor drainage and is a major determinant ofIOP. When canal resistance increases with age or under pathologicalsituation, IOP is elevated leading to glaucoma with irreversible opticnerve damage and vision loss. It is therefore an important target forglaucoma therapy. Recently, we provided the first evidence thatSchlemm's canal expresses Prox-1, the master control gene of lymphaticformation (Truong T N, Li H, Hong Y K, Chen L. Novel characterizationand live imaging of Schlemm's canal expressing Prox-1. PLoS One. 2014;9(5):e98245).

Wnt5a belongs to the Wnt family that comprises ligands and receptors inmammals.

Here we disclose that Wnt5a is expressed on Schlemm's canal, and itsexpression is regulated in response to sheer stress change. Moreover, byinhibiting Wnt5a, we can effectively lower IOP in vivo.

SUMMARY OF THE INVENTION

The invention provides methods and compositions for locally treatingglaucoma or pathogenic intraocular pressure.

In an aspect the invention provides a method of treating glaucoma orpathogenic intraocular pressure, comprising locally administering to aneye in need thereof a Wnt5a inhibitor.

In embodiments:

-   -   the administering step comprises delivery by eye drop or by        intracameral, subconjunctival injection, or intravitreal        injection;    -   the inhibitor is selected from an antibody, an siRNA, an small        interfering peptide, and a small molecule inhibitor;    -   the inhibitor is delivered by viral vector, such as AAV or        lentivirus; and/or    -   the administration is topical, and the inhibitor is administered        in form of a topical ophthalmic gel, ointment, suspension or        solution;

In another aspect, the invention provides an ophthalmic formulation of aWnt5a-specific inhibitor selected from an antibody, an siRNA, an smallinterfering peptide, and a small molecule inhibitor, in unit dosage formfor treating glaucoma or pathogenic intraocular pressure.

In embodiments:

-   -   the formulation is in the form of a topical ophthalmic gel,        ointment, suspension or solution, such as an ophthalmic        lubricant;    -   the dosage form is an inhibitor-loaded contact lens, eye drop,        depot or bollus    -   the formulation is packaged in an eye drop dispenser;    -   the formulation is loaded in a syringe configured for        intracameral injection, subconjuctival injection or intravitreal        injection; and/or    -   the formulation further comprises excipients and/or features        suitable for direct, topical delivery to the eye, such as        selected from the group consisting of ophthalmically suitable        clarity, pH buffer, tonicity, viscosity, stability and        sterility.

The invention encompasses all combinations of the particular embodimentsrecited herein. The methods may be practiced with all disclosedcompositions including specific embodiments.

DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION

The examples and embodiments described herein are for illustrativepurposes and various modifications or changes in light thereof will beapparent to persons skilled in the art and are to be included withinthis invention. Those skilled in the art will recognize a variety ofnoncritical parameters that could be changed or modified to yieldessentially similar results. The invention may exclude or be practicedin the absence of any compound, component, element or step which is notdisclosed are required herein. Unless contraindicated or notedotherwise, in these descriptions and throughout this specification, theterms “a” and “an” mean one or more. All publications, patents, andpatent applications cited herein, including citations therein, arehereby incorporated by reference in their entirety for all purposes.

The disclosed Wnt5a inhibition methods can be genetic manipulation,and/or administrations of small interfering RNAs (siRNAs), antibodies,small molecules, etc, many of which are commercially available fromsources like Applied Biological Materials Inc. (ABM, Richmond BC), LifeTechnologies (ThermoFisher Scientific), Sigma-Aldrich, etc. The methodscan be used alone to lower intraocular pressure and to prevent or treatglaucoma, and/or in combination with other therapeutic approaches, suchas eye drops, medications, laser, implanted devices, and surgery, etc.to prevent or treat glaucoma.

Prototypical Examples

Wnt5a is expressed on human primary SC cells in culture and mouse SC invivo. Wnt5a expression is regulated with sheer stress change, asanalyzed by quantitative real-time PCR assay. We also demonstrate thatWnt5a expression in human SC cells can be down-regulated byWnt5a-specific siRNA, which affects SC cell functions as well. InSC-specific Wnt5a gene conditional knockout mice IOP elevation inducedin a glaucoma model is significantly reduced compared to controllittermates. No significant difference was found in baseline IOP betweenthe knockout mice and control littermates. Compared with the controllittermates that had IOP elevation at all time-points studied, Wnt5aknockout mice only showed elevated IOP at the early (within 24 hours)but not later time points, indicating an unsustainable IOP increase withWnt5a intervention. We also demonstrate that wnt5a intervention iseffective in protecting retinal nerve fiber layer and increasing SCpermeability, a target for enhancement of aqueous movement through theconventional outflow system to manage ocular hypertension (e.g. Tam etal., Scientific Reports 7:40717, DOI: 10.1038/srep40717). Theseexperiments demonstrate Wnt5a is an effective therapeutic target forglaucoma management. These results are further demonstrated by selectiveinhibition of Wnt5a by CRISPR gene editing employing the methods ofHuang, et al. (Nature Communications, 2017; 8 (1) DOI:10.1038/s41467-017-00140-3).

We next developed experimental protocols to demonstrate efficacy ofWnt5a siRNA inhibitor treatments to reduce IOP. For these protocolsWnt5a specific siRNA was obtained commercially (human WNTSA siRNA, LifeTechnologies; Anastas, et al. J. Clin. Investig. 2014, 124, 2877-2890).In one protocol subconjuctival injection of siRNA is performed asdescribed by Yuen et al. (2014, Invest Ophthalmol Vis Sci. 2014;55:3320-3327). Mice are randomly selected to receive subconjunctivalinjection of 5 uL (0.2 lg/uL) siRNA or control twice a week for 2 weeks.In a second protocol intracameral injection of siRNA is performed asdescribed by Tam et al. (2017, Scientific Reports 7, 40717). Mice areanaesthetized by intra-peritoneal injection, and pupils are dilated. Apulled blunt-ended micro-glass needle is first used to puncture thecornea to withdraw aqueous humour Immediately after puncture, a pulledblunt-ended micro-glass needle attached to a 10 μl syringe is insertedthrough the puncture, and 1.5 μl of PBS containing 1 μg siRNA isadministered into the anterior chamber. Contralateral eyes receive anidentical injection of 1.5 μl containing the same concentration ofscrambled siRNA. These experiments demonstrate that Wnt5a-specificinhibitor siRNA delivered locally by either subconjuctival injection orintracameral injection is an effective therapy for pathogenic IOP.

To assess the effect of siRNA delivered by eye drops on IOP, wedeveloped an additional protocol based on the methods of Martinez et al.(Mol Ther. 2014 January; 22(1):81-91), wherein New Zealand White rabbitsreceive a topical administration of either 20 nmol/day of siRNA orphosphate-buffered saline (PBS) over a period of 4 consecutive days.Treated eyes present a significant IOP decrease when compared with thevehicle-treated group. The effect of the siRNA on IOP is detectable 2days after the first administration and values remains below basallevels until ˜2 days after the last administration. We also adapted anoral water overloading model in New Zealand White Rabbits to evaluatethe IOP-lowering effect of Wnt5a siRNA in pathologic conditions likeobserved in glaucoma. Initially four different doses of siRNA (10 nmol,20 nmol, 40 nmol, and 60 nmol/eye/day) are administered a total of threetimes: 48, 24, and 2 hours before hypertension induction. All treatmentsare applied in both eyes and IOP measured before hypertension inductionand every 20 minutes up to 120 minutes after oral overloading. Analysisof the results shows that the Wnt5a siRNA provides significantprotection against the rise of IOP at all doses tested.

To confirm the efficacy and specificity of Wnt5a siRNA on IOP, a largergroup of animals is treated with a dose of 40 nmol/eye/day over a periodof 4 consecutive days; on the fourth day, ocular hypertension is inducedby water loading. Control results demonstrate that water loading causedan increase in IOP during the first hour after hypertension induction inanimals treated with PBS. Analysis performed by comparing IOP values ateach time point indicate that treatment with siRNA significantly reducedΔIOP values within the first hour compared with PBS-treated animals. Theeffect is specific since treatment with a scrambled sequence siRNA hasno effect on IOP.

We next developed experimental protocols to demonstrate efficacy ofWnt5a specific antibody inhibitor treatments to reduce IOP. Theseprotocols employ two different antibodies: anti-human WNTSA antibodyproduced in rabbit purified immunoglobulin, buffered aqueous solution(Sigma-Aldrich SAB1411396), and an anti-human WNTSA monoclonal antibodyproduced in mouse clone 6F2, ascites fluid (Sigma-Aldrich SAB5300183),although other Wnt5a antibodies can be used, e.g. Hanaki et al., MolCancer Ther 11(2) February 2012; He et al. Oncogene. 2005, 24 (18):3054-3058. Using both the mouse and rabbit models (supra), theseexperiments demonstrate that Wnt5a-specific antibody inhibitor deliveredlocally by eye drops is an effective therapy for pathogenic IOP.

In an exemplary model system intraocular hypertension was induced in theright eye (OD) of wildtype normal mice and Wnt5a neutralizing antibodieswere administered to assess their therapeutic effects on IOP and otherparameters of glaucoma including corneal edema, retinal ganglion cell(RGC) death, and RNFL thinning Compared to the control group where IOPwas significantly elevated in the right eyes of the mice, IOP in Wnt5aantibody treated eyes was significantly lower and maintained at thebaseline level. Wnt5a intervention reduced corneal edema, as measured bycentral corneal thickness in vivo by OCT. Increased corneal thicknesswas observed in the control group after IOP was increased, but not inWnt5a antibody treated eyes. Wnt5a intervention reduced RGC death aswell as RNFL thinning in the treated eyes. These were detected byimmunostaining and OCT, respectively. These results confirmed thattopical Wnt5a antibody intervention significantly lowers IOP andprotects the cornea and retina in a mouse model of glaucoma.

We next designed experimental protocols to demonstrate efficacy of Wnt5aspecific antagonist peptide and small molecule inhibitor treatments toreduce IOP. These protocols employ a t-butyloxycarbonyl-modifiedWnt5a-derived hexapeptide (Box5) that functions as a potent antagonistof Wnt5a (Jenei, et la., PNAS USA, 106 (46), 19473-8), and6,7-dihydro-10alpha-hydroxy radicicol, a potent WNT-5A expressioninhibitor with relatively low toxicity and excellent stability(Shinonaga et al. Bioorg Med Chem. 2009 Jul. 1; 17(13):4622-35). Againusing both the mouse and rabbit models (supra), these experimentsdemonstrate that Wnt5a-specific modified peptide inhibitor and smallmolecule inhibitor of Wnt5a expression, delivered locally by eye dropsare effective therapies for pathogenic IOP.

1. A method of treating glaucoma or pathogenic intraocular pressure,comprising locally administering to an eye in need thereof a Wnt5ainhibitor.
 2. The method of claim 1 wherein the administering stepcomprises delivery by eye drop or by intracameral, subconjuctivalinjection or intravitreal injection.
 3. The method of claim 1 whereinthe administration is topical, and the inhibitor is administered in formof a topical ophthalmic gel, ointment, suspension or solution.
 4. Themethod of claim 1 wherein the inhibitor is selected from an antibody, ansiRNA, an small interfering peptide, and a small molecule inhibitor. 5.The method of claim 2 wherein the inhibitor is selected from anantibody, an siRNA, an small interfering peptide, and a small moleculeinhibitor.
 6. The method of claim 3 wherein the inhibitor is selectedfrom an antibody, an siRNA, an small interfering peptide, and a smallmolecule inhibitor.
 7. The method of claim 1 wherein the inhibitor is ananti-Wnt5a antibody.
 8. The method of claim 1 wherein the inhibitor isan anti-Wnt5a siRNA
 9. The method of claim 1 wherein the inhibitor is ananti-Wnt5a small interfering peptide
 10. The method of claim 1 whereinthe inhibitor is an anti-Wnt5a small molecule inhibitor selected fromt-butyloxycarbonyl-modified Wnt5A hexapeptide (Box5) and6,7-dihydro-10alpha-hydroxy radicicol.
 11. The method of claim 1 furthercomprising detecting a resultant decrease in the eye of a parameter ofglaucoma selected from pathogenic intraocular pressure (IOP), cornealedema, retinal ganglion cell (RGC) death, and retinal nerve fiber layer(RNFL) thinning Para
 31. 12. The method of claim 2 further comprisingdetecting a resultant decrease in the eye of a parameter of glaucomaselected from pathogenic intraocular pressure (IOP), corneal edema,retinal ganglion cell (RGC) death, and retinal nerve fiber layer (RNFL)thinning.
 13. The method of claim 3 further comprising detecting aresultant decrease in the eye of a parameter of glaucoma selected frompathogenic intraocular pressure (IOP), corneal edema, retinal ganglioncell (RGC) death, and retinal nerve fiber layer (RNFL) thinning.
 14. Themethod of claim 4 further comprising detecting a resultant decrease inthe eye of a parameter of glaucoma selected from pathogenic intraocularpressure (IOP), corneal edema, retinal ganglion cell (RGC) death, andretinal nerve fiber layer (RNFL) thinning.
 15. The method of claim 5further comprising detecting a resultant decrease in the eye of aparameter of glaucoma selected from pathogenic intraocular pressure(IOP), corneal edema, retinal ganglion cell (RGC) death, and retinalnerve fiber layer (RNFL) thinning.
 16. The method of claim 6 furthercomprising detecting a resultant decrease in the eye of a parameter ofglaucoma selected from pathogenic intraocular pressure (IOP), cornealedema, retinal ganglion cell (RGC) death, and retinal nerve fiber layer(RNFL) thinning.
 17. The method of claim 7 further comprising detectinga resultant decrease in the eye of a parameter of glaucoma selected frompathogenic intraocular pressure (IOP), corneal edema, retinal ganglioncell (RGC) death, and retinal nerve fiber layer (RNFL) thinning.
 18. Themethod of claim 8 further comprising detecting a resultant decrease inthe eye of a parameter of glaucoma selected from pathogenic intraocularpressure (IOP), corneal edema, retinal ganglion cell (RGC) death, andretinal nerve fiber layer (RNFL) thinning.
 19. The method of claim 9further comprising detecting a resultant decrease in the eye of aparameter of glaucoma selected from pathogenic intraocular pressure(IOP), corneal edema, retinal ganglion cell (RGC) death, and retinalnerve fiber layer (RNFL) thinning.
 20. The method of claim 10 furthercomprising detecting a resultant decrease in the eye of a parameter ofglaucoma selected from pathogenic intraocular pressure (IOP), cornealedema, retinal ganglion cell (RGC) death, and retinal nerve fiber layer(RNFL) thinning.
 21. An ophthalmic formulation of a Wnt5a-specificinhibitor selected from an antibody, an siRNA, an small interferingpeptide, and a small molecule inhibitor, in unit dosage form fortreating glaucoma or pathogenic intraocular pressure, whereinoptionally: in the form of a topical ophthalmic gel, ointment,suspension or solution; the dosage form is an inhibitor-loaded contactlens, eye drop, depot or bollus; packaged in an eye drop dispenser;loaded in a syringe configured for intracameral injection,subconjuctival injection or intravitreal injection; and/or furthercomprising excipients and features suitable for direct, topical deliveryto the eye, selected from the group consisting of opthalmically suitableclarity, pH buffer, tonicity, viscosity, stability and sterility.